DOCSLIB.ORG

The Small Gtpase Rac1 Contributes to Extinction of Aversive Memories

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Small Gtpase Rac1 Contributes to Extinction of Aversive Memories 7096 • The Journal of Neuroscience, July 26, 2017 • 37(30):7096–7110 Behavioral/Cognitive The Small GTPase Rac1 Contributes to Extinction of Aversive Memories of Drug Withdrawal by Facilitating GABAA Receptor Endocytosis in the vmPFC Weisheng Wang,1* Yun-Yue Ju,1* Qi-Xin Zhou,2* Jian-Xin Tang,3 Meng Li,2 Lei Zhang,4 Shuo Kang,1 Zhong-Guo Chen,1 Yu-Jun Wang,1 Hui Ji,3 XYu-Qiang Ding,4 XLin Xu,2,5,6 and Jing-Gen Liu1 1Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Collaborative Innovation Center for Brain Science, Shanghai 201203, China, 2Key Laboratory of Animal Models and Human Disease Mechanisms, and Laboratory of Learning and Memory, Kunming Institute of Zoology, Chinese Academy of Science, Kunming 650223, China, 3Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, China, 4Department of Anatomy and Neurobiology, Collaborative Innovation Center for Brain Science, Tongji University School of Medicine, Shanghai 200092, China, 5KIZ-SU Joint Laboratory of Animal Model and Drug Development, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China, and 6CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai 200031, China Extinction of aversive memories has been a major concern in neuropsychiatric disorders, such as anxiety disorders and drug addiction. However, the mechanisms underlying extinction of aversive memories are not fully understood. Here, we report that extinction of conditionedplaceaversion(CPA)tonaloxone-precipitatedopiatewithdrawalinmaleratsactivatesRhoGTPaseRac1intheventromedial prefrontal cortex (vmPFC) in a BDNF-dependent manner, which determines GABAA receptor (GABAAR) endocytosis via triggering synaptic translocation of activity-regulated cytoskeleton-associated protein (Arc) through facilitating actin polymerization. Active Rac1 is essential and sufficient for GABAAR endocytosis and CPA extinction. Knockdown of Rac1 expression within the vmPFC of rats using Rac1-shRNAsuppressedGABAARendocytosisandCPAextinction,whereasexpressionofaconstitutivelyactiveformofRac1accelerated GABAAR endocytosis and CPA extinction. The crucial role of GABAAR endocytosis in the LTP induction and CPA extinction is evinced by the findings that blockade of GABAAR endocytosis by a dynamin function-blocking peptide (Myr-P4) abolishes LTP induction and CPA extinction. Thus, the present study provides first evidence that Rac1-dependent GABAAR endocytosis plays a crucial role in extinction of aversive memories and reveals the sequence of molecular events that contribute to learning experience modulation of synaptic GABAAR endocytosis. Key words: aversive memory; drug withdrawal; endocytosis; extinction; GABAA receptor; Rac1 Significance Statement This study reveals that Rac1-dependent GABAAR endocytosis plays a crucial role in extinction of aversive memories associated with drug withdrawal and identifies Arc as a downstream effector of Rac1 regulations of synaptic plasticity as well as learning and memory, thereby suggesting therapeutic targets to promote extinction of the unwanted memories. Introduction drug taking (Koob, 2000; Hutcheson et al., 2001). Extinction of The aversive memory associated with drug withdrawal can evoke such memory has been proposed as a therapeutic strategy for the motivational and/or emotional states that lead to compulsive treatment of drug addiction (Barad, 2005; Davis et al., 2006; Hof- mann et al., 2006). Our previous study demonstrates that epigenetic regulation within the ventromedial prefrontal cortex (vmPFC) of Received Dec. 19, 2016; revised June 1, 2017; accepted June 8, 2017. Author contributions: W.W., Y.-Y.J., and Y.-Q.D. designed research; W.W., Y.-Y.J., Q.-X.Z., J.-X.T., M.L., L.Z., S.K., Z.-G.C., Y.-J.W., and H.J. performed research; W.W. and Y.-Y.J. analyzed data; L.X. and J.-G.L. wrote the paper. Correspondence should be addressed to either of the following: Dr. Lin Xu, Key Laboratory of Animal Models and This work was supported by Ministry of Science and Technology of China Grants 2013CB835100 and Human Disease Mechanisms, and Laboratory of Learning and Memory, Kunming Institute of Zoology, Chinese 2015CB553502 to J.-G.L., Ministry of Science and Technology of China Grant 2013CB8351003 to L.X., Foundation of Academy of Science, Kunming 650223, China, E-mail: [email protected]; or Dr. Jing-Gen Liu, Key Laboratory of National Natural Science of China Grants 81130087 and 91232716 to J.-G.L., and Committee of Science and Tech- ReceptorResearch,ShanghaiInstituteofMateriaMedica,ChineseAcademyofSciencesandCollaborativeInnovation nology of Shanghai Grant 13JC140680 to J.-G.L. Center for Brain Science, Shanghai 201203, China, E-mail: [email protected]. The authors declare no competing financial interests. DOI:10.1523/JNEUROSCI.3859-16.2017 *W.W., Y.-Y.J., and Q.-X.Z. contributed equally to this work. Copyright © 2017 the authors 0270-6474/17/377096-15$15.00/0 Wang et al. • Small GTPase Rac1 and Extinction of Aversive Memories of Drug Withdrawal J. Neurosci., July 26, 2017 • 37(30):7096–7110 • 7097 BDNF transcription is required for the extinction of conditioned aptic plasticity and learning and reveal a mechanism of extinction place aversion (CPA) to naloxone-precipitated opiate withdrawal of drug withdrawal memory. (Wang et al., 2012). However, the mechanisms by which BDNF in the mPFC contribute to the extinction of aversive memory are unknown. Materials and Methods Extinction of classical fear conditioning is an active learning Animals Sprague Dawley male rats weighing 220–250 g were obtained from the process requiring synaptic structural plasticity (Lai et al., 2012). Laboratory Animal Center, the Chinese Academy of Sciences, or Kun- Dendritic spines, which are central to synaptic structural plastic- ming Medical University. Rats were housed 3 per cage and maintained on ity (Lu¨scher et al., 2000), undergo activity-dependent structural a 12 h light/dark cycle with access to food and water ad libitum. Rac1 remodeling that has been proposed to be a cellular basis of LTP transgenic mice were purchased from The Jackson Laboratory (stock (Engert and Bonhoeffer, 1999; Toni et al., 1999) as well as learn- #012361) and reproduced and bred until 2 months old at the Animal ing and memory (Matsuzaki et al., 2004; Yang et al., 2009; Fu et Center of TongJi University. All experimental procedures were in strict al., 2012). Activity-dependent changes in spine structure rely accordance with the National Institutes of Health Guide for the care and on rearrangements of the actin cytoskeleton. One of the best- use of laboratory animals. characterized pathways for regulation of actin dynamics involves the Rho family of small GTPases, Rac1, Cdc42, and Rho (Hall, Drugs and antibodies 1998, 2005). Rac1 has been shown to play a crucial role in synapse Morphine hydrochloride was purchased from Qinghai Pharmaceutical General Factory with a permission license for its use in experiments from formation and plasticity (Luo, 2000; Nakayama et al., 2000). local government (Shanghai and Kunming). Naloxone hydrochloride Studies of the role of Rac1 in spine morphogenesis in vivo and was supplied by Sigma-Aldrich (catalog #N7758). Latrunculin A was vitro have suggested that Rac1 plays an important role in regulat- obtained from Merck/Millipore (catalog #428021) and dissolved in 25% ing the size and density of spines in neurons (Luo et al., 1996; DMSO to a final concentration of 0.5 ␮g/␮l; BDNF was obtained from Zhang et al., 2003; Tolias et al., 2005; Xie et al., 2007). Although R&D Systems (catalog #248-BD-025/CF) and dissolved in PBS to 1.5 substantial evidence demonstrates that Rac1 is essential for di- ␮g/␮l; TrkB/FC was obtained from Sigma-Aldrich (catalog #T8694) and verse forms of learning, little is known whether Rac1 participates dissolved in PBS to 1.3 ␮g/␮l; NSC23766 (catalog #2161), Myr-P4 (cat- in an opposing form of learning (extinction) and how Rac1 con- alog #1776), and Myr-S (control) were obtained from Tocris Bioscience ␮ ␮ ␮ ␮ tributes to extinction of an established memory. and dissolved in PBS to 10 g/ l, 60 pmol/ l, and 60 pmol/ l, respec- Information processing in brain is controlled by a dynamic tively. The antibodies of anti-BDNF (catalog #sc-546, RRID: AB_630940) and anti-Arc/Arg3.1 (catalog #sc-17839, RRID: AB_626696) were pur- interaction between excitatory and inhibitory neurotransmis- chased from Santa Cruz Biotechnology. The antibodies of anti-pPak1 (cat- sion. GAGAA receptors (GABAARs) constitute the major inhibi- alog #2601S, RRID: AB_330220), anti-pCofilin (catalog #3311S, RRID: tory synaptic transmission network in the brain and are essential AB_330238), and anti-Rac1/cdc42 (catalog #4651S, RRID: AB_10612265) for maintaining the excitatory/inhibitory balance of neuronal cir- were purchased from Cell Signaling Technology. The antibodies of anti- ␤ cuits (Lu¨scher et al., 2011). Modulating the strength of GABAergic GABAA 3 (catalog #05-474, RRID: AB_11212228) were purchased from inhibitory transmission has important implications for synaptic Merck/Millipore. The antibodies of anti-actin (catalog #A5441, RRID: plasticity and information processing in the brain (Smith and AB_476744) were purchased from Sigma-Aldrich. Kittler, 2010). It has been shown that inhibiting GABAergic transmission facilitates LTP induction in excitatory synapses in Intra-vmPFC microinjection many brain regions, including mPFC (Wigstro¨m and Gustafsson, Surgery. Rats (weighing 220–280 g when surgery
Load more

Recommended publications
  • Dynamin Functions and Ligands: Classical Mechanisms Behind
    1521-0111/91/2/123–134$25.00 http://dx.doi.org/10.1124/mol.116.105064 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:123–134, February 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics MINIREVIEW Dynamin Functions and Ligands: Classical Mechanisms Behind Mahaveer Singh, Hemant R. Jadhav, and Tanya Bhatt Department of Pharmacy, Birla Institute of Technology and Sciences Pilani, Pilani Campus, Rajasthan, India Received May 5, 2016; accepted November 17, 2016 Downloaded from ABSTRACT Dynamin is a GTPase that plays a vital role in clathrin-dependent pathophysiology of various disorders, such as Alzheimer’s disease, endocytosis and other vesicular trafficking processes by acting Parkinson’s disease, Huntington’s disease, Charcot-Marie-Tooth as a pair of molecular scissors for newly formed vesicles originating disease, heart failure, schizophrenia, epilepsy, cancer, dominant ’ from the plasma membrane. Dynamins and related proteins are optic atrophy, osteoporosis, and Down s syndrome. This review is molpharm.aspetjournals.org important components for the cleavage of clathrin-coated vesicles, an attempt to illustrate the dynamin-related mechanisms involved phagosomes, and mitochondria. These proteins help in organelle in the above-mentioned disorders and to help medicinal chemists division, viral resistance, and mitochondrial fusion/fission. Dys- to design novel dynamin ligands, which could be useful in the function and mutations in dynamin have been implicated in the treatment of dynamin-related disorders. Introduction GTP hydrolysis–dependent conformational change of GTPase dynamin assists in membrane fission, leading to the generation Dynamins were originally discovered in the brain and identi- of endocytic vesicles (Praefcke and McMahon, 2004; Ferguson at ASPET Journals on September 23, 2021 fied as microtubule binding partners.
    [Show full text]
  • Crystal Structure of a Small G Protein in Complex with the Gtpase
    letters to nature of apolipoproteins2,12–14. However, our results show that the residues lipoprotein metabolism involving cell surface heparan sulfate proteoglycans. J. Biol. Chem. 269, 2764– 2+ 2772 (1994). in the conserved acidic motif of LR5 are buried to participate in Ca 16. Innerarity, T. L., Pitas, R. E. & Mahley, R. W. Binding of arginine-rich (E) apoprotein after coordination, instead of being exposed on the surface of the domain recombination with phospholipid vesicles to the low density lipoprotein receptors of fibroblasts. J. Biol. Chem. 254, 4186–4190 (1979). (Fig. 4a). Although lipoprotein uptake by members of the LDLR 17. Otwinowski, Z. & Minor, W. Data Collection and Processing (eds Sawyer, L., Isaacs, N. & Bailey, S.) family may involve an electrostatic component, perhaps through 556–562 (SERC Daresbury Laboratory, Warrington, UK, 1993). association of the lipoproteins with cell-surface proteoglycans15, the 18. CCP4. The SERC (UK) Collaborative Computing Project No. 4 A Suite of Programs for Protein Crystallography (SERC Daresbury Laboratory, Warrington, UK, 1979). LR5 structure demonstrates that the primary role for the conserved 19. Cowtan, K. D. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34–38 (1994). acidic residues in LDL-A modules is structural. It has been noted 20. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 previously that only lipid-associated apolipoproteins bind with (1991). 16 high affinity to the LDLR ; the LR5 structure suggests an alternative 21.
    [Show full text]
  • Ras Gtpase Chemi ELISA Kit Catalog No
    Ras GTPase Chemi ELISA Kit Catalog No. 52097 (Version B3) Active Motif North America 1914 Palomar Oaks Way, Suite 150 Carlsbad, California 92008, USA Toll free: 877 222 9543 Telephone: 760 431 1263 Fax: 760 431 1351 Active Motif Europe Waterloo Atrium Drève Richelle 167 – boîte 4 BE-1410 Waterloo, Belgium UK Free Phone: 0800 169 31 47 France Free Phone: 0800 90 99 79 Germany Free Phone: 0800 181 99 10 Telephone: +32 (0)2 653 0001 Fax: +32 (0)2 653 0050 Active Motif Japan Azuma Bldg, 7th Floor 2-21 Ageba-Cho, Shinjuku-Ku Tokyo, 162-0824, Japan Telephone: +81 3 5225 3638 Fax: +81 3 5261 8733 Active Motif China 787 Kangqiao Road Building 10, Suite 202, Pudong District Shanghai, 201315, China Telephone: (86)-21-20926090 Hotline: 400-018-8123 Copyright 2021 Active Motif, Inc. www.activemotif.com Information in this manual is subject to change without notice and does not constitute a commit- ment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any kind, either explicit or implied. Information may be changed or updated in this manual at any time. This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, in whole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and interna- tional treaties. The manufacturer of this documentation is Active Motif, Inc. © 2021 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008.
    [Show full text]
  • Plasma Membrane Repairs by Small Gtpase Rab3a
    Published June 20, 2016 JCB: SpotlightReview Plasma membrane repairs by small GTPase Rab3a Camilla Raiborg1,2 and Harald Stenmark1,2 1Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo, Norway 2Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway Lysosomes fuse with the plasma membrane to help repair in lysosome-mediated PM repair has not been addressed. In this membrane lesions, but how they are positioned close to issue, Encarnação et al. screened siRNAs that target human Rab GTPases to identify Rabs involved in translocating lysosomes to these lesions is not fully understood. Now, Encarnação et the PM in response to the Ca2+ ionophore ionomycin and in the al. (2016. J. Cell Biol. http ://dx .doi .org /10 .1083 /jcb repair of PM holes caused by the bacterial toxin Streptolysin-O .201511093) demonstrate that the lysosomal GTPase (Encarnação et al., 2016). One of the most prominent hits in this Rab3a and its effectors orchestrate lysosome positioning screen was Rab3a, a GTPase previously characterized mainly in the regulation of Ca2+-induced exocytosis of synaptic vesicles and plasma membrane repair. in neurons and of dense-core vesicles in endocrine cells (Gep- pert et al., 1994; Johannes et al., 1994). Even though Rab3a is Downloaded from When the plasma membrane (PM) of eukaryotic cells is predominantly expressed in neurons and endocrine tissues, En- wounded by mechanical damage or by bacterial toxins, the carnação et al. (2016) were able to detect its expression in HeLa wound is normally repaired within seconds by mechanisms cells and melanocytes.
    [Show full text]
  • G Protein Regulation of MAPK Networks
    Oncogene (2007) 26, 3122–3142 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW G Protein regulation of MAPK networks ZG Goldsmith and DN Dhanasekaran Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA G proteins provide signal-coupling mechanisms to hepta- the a-subunits has been used as a basis for the helical cell surface receptors and are criticallyinvolved classification of G proteins into Gs,Gi,Gq and G12 in the regulation of different mitogen-activated protein families in which the a-subunits that show more than kinase (MAPK) networks. The four classes of G proteins, 50% homology are grouped together (Simon et al., defined bythe G s,Gi,Gq and G12 families, regulate 1991). In G-protein-coupled receptor (GPCR)-mediated ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by signaling pathways, ligand-activated receptors catalyse different mechanisms. The a- as well as bc-subunits are the exchange of the bound GDP to GTP in the a-subunit involved in the regulation of these MAPK modules in a following which the GTP-bound a-subunit disassociate context-specific manner. While the a- and bc-subunits from the receptor as well as the bg-subunit. The GTP- primarilyregulate the MAPK pathwaysvia their respec- bound a-subunit and the bg-subunit stimulate distinct tive effector-mediated signaling pathways, recent studies downstream effectors including enzymes, ion channels have unraveled several novel signaling intermediates and small GTPase, thus regulating multiple signaling including receptor tyrosine kinases and small GTPases pathways including those involved in the activation of through which these G-protein subunits positivelyas well mitogen-activated protein kinase (MAPK) modules as negativelyregulate specific MAPK modules.
    [Show full text]
  • Uncoupling of Dynamin Polymerization and Gtpase Activity Revealed By
    RESEARCH ARTICLE Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab Valentina Galli1, Rafael Sebastian2, Sandrine Moutel3,4, Jason Ecard3, Franck Perez3, Aure´ lien Roux1,5* 1Department of Biochemistry, University of Geneva, Geneva, Switzerland; 2Department of Computer Sciences, Universidad de Valencia, Valencia, Spain; 3Institut Curie, PSL Research University, Paris, France; 4Translational Department, Institut Curie, Paris, France; 5Swiss National Centre for Competence in Research Programme Chemical Biology, Geneva, Switzerland Abstract Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction. DOI: https://doi.org/10.7554/eLife.25197.001 *For correspondence: [email protected] Competing interests: The Introduction authors declare that no There are three mammalian variants of dynamin that share 80% sequence identity.
    [Show full text]
  • Small Gtpases of the Ras and Rho Families Switch On/Off Signaling
    International Journal of Molecular Sciences Review Small GTPases of the Ras and Rho Families Switch on/off Signaling Pathways in Neurodegenerative Diseases Alazne Arrazola Sastre 1,2, Miriam Luque Montoro 1, Patricia Gálvez-Martín 3,4 , Hadriano M Lacerda 5, Alejandro Lucia 6,7, Francisco Llavero 1,6,* and José Luis Zugaza 1,2,8,* 1 Achucarro Basque Center for Neuroscience, Science Park of the Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), 48940 Leioa, Spain; [email protected] (A.A.S.); [email protected] (M.L.M.) 2 Department of Genetics, Physical Anthropology, and Animal Physiology, Faculty of Science and Technology, UPV/EHU, 48940 Leioa, Spain 3 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, 180041 Granada, Spain; [email protected] 4 R&D Human Health, Bioibérica S.A.U., 08950 Barcelona, Spain 5 Three R Labs, Science Park of the UPV/EHU, 48940 Leioa, Spain; [email protected] 6 Faculty of Sport Science, European University of Madrid, 28670 Madrid, Spain; [email protected] 7 Research Institute of the Hospital 12 de Octubre (i+12), 28041 Madrid, Spain 8 IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain * Correspondence: [email protected] (F.L.); [email protected] (J.L.Z.) Received: 25 July 2020; Accepted: 29 August 2020; Published: 31 August 2020 Abstract: Small guanosine triphosphatases (GTPases) of the Ras superfamily are key regulators of many key cellular events such as proliferation, differentiation, cell cycle regulation, migration, or apoptosis. To control these biological responses, GTPases activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some small GTPases also guanine nucleotide dissociation inhibitors (GDIs).
    [Show full text]
  • Small G-Protein They Bind GTP, and “OFF” After the GTP Has Been Hydrolyzed to GDP (Which in Turn Remains Bound)
    MONOMERIC G-PROTEINS (GTPases) In addition to heterotrimeric G-proteins, there is a superfamily of small monomeric G-proteins. Their size is around 21 kDa, approximately one half of the average size of the subunits of heterotrimeric proteins. These small proteins also operate as switches, which are “ON” when Small G-protein they bind GTP, and “OFF” after the GTP has been hydrolyzed to GDP (which in turn remains bound). There is a larger family of small GTP-binding switch proteins, related to G Monomeric G-proteins are activated by proteins which induce a conformational change resulting in reduced affinity to GDP, and thus in G-protein : heterotrimeric G proteins GDP release. The general term for such proteins is GEF (guanine nucleotide exchange factor). The GEFs of the small G-proteins are not the activated receptors, but rather proteins downstream the signaling cascade. GEFs vary with regard to their mode of activation and their selectivity for specific monomeric G-proteins. Small GTP-binding proteins include (roles indicated): initiation & elongation factors (protein synthesis). Compared to the subunit of heterotrimeric G-proteins, GTPase activity Ras (growth factor signal cascades). of the monomeric G-proteins is very low in the absence of interference. Rab (vesicle targeting and fusion). However, association with a protein of the GAP (GTPase-activating ARF (forming vesicle coatomer coats). protein) type results in very rapid GTP hydrolysis. Ran (transport of proteins into & out of the nucleus). As in the case of GEFs, the activation state of GAPs can be regulated, and Rho (regulation of actin cytoskeleton) GAPs are selective with regard to the small G-proteins they affect.
    [Show full text]
  • Spatiotemporal Imaging of Small Gtpases Activity in Live Cells
    Spatiotemporal imaging of small GTPases activity in live cells Stephanie Vossa,b, Dennis M. Krügera, Oliver Kochc, and Yao-Wen Wua,b,1 aChemical Genomics Centre, Max Planck Society, 44227 Dortmund, Germany; bMax-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany; and cFaculty of Chemistry and Chemical Biology, TU Dortmund University, 44227 Dortmund, Germany Edited by Jennifer Lippincott-Schwartz, Howard Hughes Medical Institute, Ashburn, VA, and approved October 31, 2016 (received for review August 22, 2016) Ras-like small GTPases function as molecular switches and regulate intermolecular (17) or intramolecular Förster resonance energy diverse cellular events. To examine the dynamics of signaling transfer (FRET) (e.g., Raichu sensors), as shown for H-Ras and requires spatiotemporal visualization of their activity in the cell. Rap1 (18), Rho (19), and Rab5 (20). However, quantification of Current small GTPase sensors rely on specific effector domains that FRET requires calibration of the fluorophore concentrations are available for only a small number of GTPases and compete for and undesired signals from donor emission bleed-through, endogenous regulator/effector binding. Here, we describe versa- moreover, direct acceptor excitation have to be considered. In a tile conformational sensors for GTPase activity (COSGAs) based on FRET independent approach, the CRIB domain of the effector the conserved GTPase fold. Conformational changes upon GDP/ WASP labeled with an environment-sensitive dye displayed an GTP exchange were directly observed in solution, on beads, and in increase in fluorescence intensity upon binding to the activated live cells by Förster resonance energy transfer (FRET). The COSGAs Cdc42 GTPase (21, 22). allow for monitoring of Rab1 and K-Ras activity in live cells using Crucial for these strategies is the availability of a domain that fluorescence lifetime imaging microscopy.
    [Show full text]
  • The Missing Link Between Transforming G Proteins and Rho?
    Oncogene (2001) 20, 1661 ± 1668 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho? Shigetomo Fukuhara1,2, Hiroki Chikumi1 and J Silvio Gutkind*,1 1Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland MD 20892-4340, USA Oncogene (2001) 20, 1661 ± 1668. aq, and a12 (Wilkie et al., 1992). The aq family of heterotrimeric G protein a subunits is coupled to Keywords: Rho; G proteins; Ras; signal transduction; phospholipase-C b to induce the hydrolysis of G protein-coupled receptors phosphatidylinositol bis phosphate, the consequent rise in the intracellular concentration of Ca2+, and the activation of protein kinase C, whereas the as and ai Introduction families are known to activate and inhibit adenylyl cyclase to increase and decrease the level of cyclic Many mitogens promote cell proliferation by acting AMP, respectively. ai proteins also regulate ion on cell surface receptors that either possess an channels, certain phospholipases and phosphodies- intrinsic protein-tyrosine kinase activity (Yarden et terases (Hamm, 1998). The targets for both members al., 1986) or interact with heterotrimeric GTP- of the a12 family, a12 and a13, were unknown until binding proteins (G proteins). The latter are recently. In addition, eleven-G protein g subunits and collectively known as G protein-coupled receptors ®ve G protein b subunits have been cloned so far and (GPCRs) and comprise the largest group of cell they regulate certain ion channels, phospholipase-C, surface molecules involved in signal transmission.
    [Show full text]
  • WO 2017/129998 Al 3 August 2017 (03.08.2017) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/129998 Al 3 August 2017 (03.08.2017) P O P C T (51) International Patent Classification: (72) Inventors: CARPENTER, Byron; Medical Research C07K 14/47 (2006.01) Council, Polaris House, North Star Avenue, Swindon SN2 1FL (GB). LESLIE, Andrew; Medical Research Council, (21) International Application Number: Polaris House, North Star Avenue, Swindon SN2 1FL PCT/GB20 17/050221 (GB). NEHME, Rony; Medical Research Council, Polaris (22) International Filing Date: House, North Star Avenue, Swindon SN2 1FL (GB). 27 January 2017 (27.01 .2017) TATE, Christopher Gordon; Medical Research Council, Polaris House, North Star Avenue, Swindon SN2 1FL (25) Filing Language: English (GB). WARNE, Antony; Medical Research Council, Po (26) Publication Language: English laris House, North Star Avenue, Swindon SN2 1FL (GB). (30) Priority Data: (74) Agent: PEARS, Michael; Potter Clarkson LLP, The Bel- 1601 690.9 29 January 20 16 (29.01.2016) GB grave Centre, Talbot Street, Nottingham NG1 5GG (GB). (71) Applicant: HEPTARES THERAPEUTICS LIMITED (81) Designated States (unless otherwise indicated, for every [GB/GB]; BioPark, Broadwater Road, Welwyn Garden kind of national protection available): AE, AG, AL, AM, City Hertfordshire AL7 3AX (GB). AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
    [Show full text]
  • Ribosome-Induced Tuning of GTP Hydrolysis by a Translational Gtpase
    Ribosome-induced tuning of GTP hydrolysis by a translational GTPase Cristina Maracci, Frank Peske, Ev Dannies1, Corinna Pohl, and Marina V. Rodnina2 Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany Edited* by Arieh Warshel, University of Southern California, Los Angeles, CA, and approved August 25, 2014 (received for review July 4, 2014) GTP hydrolysis by elongation factor Tu (EF-Tu), a translational Translational GTPases (trGTPases), such as bacterial elon- GTPase that delivers aminoacyl-tRNAs to the ribosome, plays gation factor Tu (EF-Tu), EF-G, initiation factor IF2, release a crucial role in decoding and translational fidelity. The basic factor RF3, SelB, and their homologs in Archaea and eukary- reaction mechanism and the way the ribosome contributes to otes, form a subclass of the GTPase superfamily. Their GTPase catalysis are a matter of debate. Here we use mutational analysis activity is stimulated by the ribosome, suggesting that the ribo- in combination with measurements of rate/pH profiles, kinetic some acts as a GAP (2, 19, 20). Common sequence signatures of solvent isotope effects, and ion dependence of GTP hydrolysis by trGTPases are the P-loop consensus motif G/AHxDxGKT and EF-Tu off and on the ribosome to dissect the reaction mechanism. — — a crucial His residue in switch II. One of the best-studied Our data suggest that contrary to current models the reaction trGTPases is EF-Tu, a factor that in the elongation phase of in free EF-Tu follows a pathway that does not involve the critical protein synthesis delivers aminoacyl-tRNA (aa-tRNA) to the A residue H84 in the switch II region.
    [Show full text]